EP0931095A2 - Nouvelles proteines humaines lim - Google Patents
Nouvelles proteines humaines limInfo
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- EP0931095A2 EP0931095A2 EP97910998A EP97910998A EP0931095A2 EP 0931095 A2 EP0931095 A2 EP 0931095A2 EP 97910998 A EP97910998 A EP 97910998A EP 97910998 A EP97910998 A EP 97910998A EP 0931095 A2 EP0931095 A2 EP 0931095A2
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- hlim
- gly
- seq
- sequence
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- NOVEL HUMAN LIM PROTEINS TECHNICAL FIELD This invention relates to nucleic acid and amino acid sequences of novel human LIM proteins and to the use of these sequences in the diagnosis, prevention, and treatment of disease.
- the LIM domain is a cysteine-rich motif which was first defined in the proteins Lin- 11 from C. elegans (Freyd, G. et al. (1990) Nature 344:876-879) , insulin gene enhancer binding protein ISL1 from rat (Karlsson, O. et al. (1990) Nature 344:879-882), and Mec-3 from C. elegans (Way, J.C. et al. (1988) Cell 54:5-16).
- the name LIM is derived from the first letter of the names of these three proteins.
- LIM domain The sequence of the LIM domain is highly conserved among proteins found in different tissues and across a variety of species, which is an indication of the functional importance of this domain (Sanchez-Garcia, I. et al. (1994) Trends Genet. 10:315-320).
- Two main classes of LIM proteins are known.
- One class consists of proteins that, like Lin-11, ISL1 and Mec-3, contain two LIM domains plus a homeodomain and are thus designated LIM-HD proteins.
- the second class of LIM proteins consists of one or more LIM domains without a homeodomain and are thus designated "LIM-only" proteins.
- a LIM domain is defined by the consensus amino acid sequence C-X 2 -C-X (17 _, 9) -H- X 2 -C-X 2 -C-X r C-X (16.20) -C-X 2 -[C, H, or D] (Wang, X. et al. (1992) J. Biol. Chem. 267:9176- 9184).
- the domain consists of two adjacent zinc-finger motifs. Two zinc ions bind to a LIM domain, one per zinc finger.
- LIM domains function as protein-binding interfaces (Schmeichel, K.L. et al. (1994) Cell 79:211-219) and thus may act as cofactors in cell signaling.
- LIM proteins exhibit oncogenic activity while other members of this diverse group may act as tumor suppressor molecules.
- the rhombotin genes (RBTN1 and RBTN2) encode LIM proteins which have been implicated in the control of the neoplastic phenotype.
- RBTN2 identified in childhood T cell acute lymphoblastic leukemia (Boehm, T. et al. (1988) EMBO J. 7:385-394), is essential for erythroid cell development.
- a homozygous null mutation in RBTN2 results in failure of yolk sac erythropoesis and embryonic death (Warren, A.J. et al. (1994) Cell 78:45-57).
- CRP is thus proposed to be a primary response gene induced as the cell transits from G 0 to G, or progresses from the S phase of the cell cycle, and is proposed to act as an oncogene.
- Muscle LIM proteins are involved in regulating cell-specific gene expression in heart and skeletal muscle.
- the expression of human MLP and the Drosophila homolog DMLP1 coincide with the differentiation of myoblasts into muscle cells.
- Overexpression of MLP in C2 myoblasts potentiates myogenic cell differentiation, whereas expression of antisense MLP RNA retards myoblast differentiation (Arber, S. et al. (1994) Cell 79:221- 231).
- Smooth muscle LIM protein from rat is expressed preferentially in aortic smooth muscle cells. Like MLP, it is a developmentally regulated nuclear protein. SmLIM mRNA levels decrease in vivo in response to vessel wall injury during periods of maximal smooth muscle proliferation (Jain, M.K. et al. (1996) J. Biol. Chem. 271 :10194-10199).
- Reversion-induced LIM (RIL) protein from rat is highly expressed in fibroblasts and is down-regulated in H-ras transformed cells. Expression of RIL is restored in phenotypic revertants derived from H-ras transformed cells. RIL is thus proposed to be involved in the maintenance of normal cell growth (Kiess, M. et al. (1995) Oncogene 10:61-68). RIL is expressed in brain, heart, testes and variety of epithelia. The pattern of RIL protein expression suggests a physiological function in epithelial cells and in postmitotic neurons of the brain.
- CLP36 protein is highly expressed in normal rat hepatocytes and is down-regulated in hypoxic hepatocytes. The relationship of this down-regulation to hypoxic injury is not understood. CLP36 is found in a wide variety of tissues, at high abundance in heart, lung and liver, moderate abundance in spleen and skeletal muscle, and at extremely low abundance in testis and brain (Wang H. et al. (1995) Gene 165:267-271).
- the present invention features three novel human LIM proteins, designated individually as HLIM-1, HLIM-2 and HLIM-3 and collectively as HLIM, and characterized as having similarity to the LIM proteins CRP and MLP from human and smLIM, CLP36 and RIL from rat.
- the invention features substantially purified HLIM proteins HLIM-1, HLIM-2, and HLIM-3 having chemical homology to the LIM proteins above and as shown in amino acid sequences SEQ ID NO:l, SEQ ID NO:3, and SEQ ID NO:5, respectively.
- polynucleotides that encode HLIM proteins HLIM-1, HLIM-2, and HLIM-3.
- the polynucleotides are the nucleotide sequences of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, respectively.
- the invention also relates to a polynucleotide sequence comprising the complement of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or variants thereof.
- the invention features polynucleotide sequences which hybridize under stringent conditions to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
- the invention additionally features nucleic acid sequences encoding polypeptides, oligonucleotides, peptide nucleic acids (PNA), fragments, portions or antisense molecules thereof, and expression vectors and host cells comprising polynucleotides that encode HLIM.
- the present invention also features antibodies which bind specifically to HLIM, and pharmaceutical compositions comprising substantially purified HLIM.
- the invention also features the use of agonists and antagonists of HLIM.
- Figures 1 A and IB show the amino acid sequence (SEQ ID NO: l ) and nucleic acid sequence (SEQ ID NO:2) of HLIM-1. The alignment was produced using MacDNASIS PROTM software (Hitachi Software Engineering Co., Ltd., San Bruno, CA).
- Figures 2A, 2B, 2C and 2D show the amino acid sequence (SEQ ID NO:3) and nucleic acid sequence (SEQ ID NO:4) of HLIM-2.
- Figures 3A, 3B, 3C and 3D show the amino acid sequence (SEQ ID NO:5) and nucleic acid sequence (SEQ ID NO:6) of HLIM-3.
- Figure 4 shows the amino acid sequence alignments among HLIM-1 (SEQ ID NO: l), human CRP (Gl 1 18161 ; SEQ ID NO:7) and MLP (Gl 1234841 ; SEQ ID NO: 8), and rat smLIM (Gl 1314351 ; SEQ ID NO:9).
- the alignment was produced using the multisequence alignment program of DNASTARTM software (DNASTAR Inc, Madison WI).
- Figures 5A and 5B shows the amino acid sequence alignments among HLIM-2 (SEQ ID NO:3), rat CLP36 (Gl 1020151 ; SEQ ID NO: 10) and rat RIL (Gl 887580; SEQ ID NO: l l).
- Figures 6A and 6B shows the amino acid sequence alignments among HLIM-3 (SEQ ID NO:5), rat proteins CLP36 (Gl 1020151 ; SEQ ID NO: 10) and rat RIL (Gl 887580; SEQ ID NO:l l).
- Figure 7 shows the hydrophobicity plot (MacDNASIS PRO software) for HLIM-1, SEQ ID NO: 1 ; the positive X axis reflects amino acid position, and the negative Y axis, hydrophobicity.
- Figure 8 shows the hydrophobicity plot for rat smLIM, SEQ ID NO:9.
- Figure 9 shows the hydrophobicity plot for HLIM-2, SEQ ID NO:3.
- Figure 10 shows the hydrophobicity plot for HLIM-3, SEQ ID NO:5.
- Figure 1 1 shows the hydrophobicity plot for rat CLP36, SEQ ID NO: 10.
- Figure 12 shows the northern analysis for SEQ ID NO:2.
- the northern analysis was produced electronically using LIFESEQTM database (Incyte Pharmaceuticals, Inc., Palo Alto, CA).
- Figures 13 A, 13B, 13C, 13D, 13E, 13F, 13G and 13H shows the northern analysis for SEQ ID NO:6.
- Nucleic acid sequence refers to an oligonucleotide, nucleotide, or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.
- amino acid sequence refers to an oligopeptide, peptide, polypeptide, or protein sequence and fragments or portions thereof, of a naturally occurring or synthetic molecule.
- amino acid sequence is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, "amino acid sequence” and like terms, such as
- polypeptide or "protein” are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
- Peptide nucleic acid refers to a molecule which comprises an oligomer to which an amino acid residue, such as lysine, and an amino group have been added. These small molecules, also designated anti-gene agents, stop transcript elongation by binding to their complementary strand of nucleic acid (Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63).
- HLIM refers to the amino acid sequences of substantially purified HLIM obtained from any species, particularly mammalian, including bovine, ovine, porcine, murine, equine, and preferably human, from any source whether natural, synthetic, semi-synthetic, or recombinant.
- Consensus refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, or which has been extended using XL-PCRTM (Perkin Elmer, Norwalk, CT) in the 5' and/or the 3' direction and resequenced, or which has been assembled from the overlapping sequences of more than one Incyte clone using the
- GELVIEWTM Fragment Assembly system (GCG, Madison, WI), or which has been both extended and assembled.
- a “variant" of HLIM refers to an amino acid sequence that is altered by one or more amino acids.
- the variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both.
- Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNASTAR software.
- a “deletion”, as used herein, refers to a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent.
- An “insertion” or “addition”, as used herein, refers to a change in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid or nucleotide residues, respectively, as compared to the naturally occurring molecule.
- substitution refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
- biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
- immunologically active refers to the capability of the natural, recombinant, or synthetic HLIM, or any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
- agonist refers to a molecule which, when bound to HLIM, causes a change in HLIM which modulates the activity of HLIM.
- Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to HLIM.
- Antagonist refers to a molecule which, when bound to HLIM, blocks the biological or immunological activity of HLIM.
- Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to HLIM.
- modulate refers to a change or an alteration in the biological activity of HLIM. Modulation may be an increase or a decrease in protein activity, a change in binding characteristics, or any other change in the biological, functional or immunological properties of HLIM.
- mimetic refers to a molecule, the structure of which is developed from knowledge of the structure of HLIM or portions thereof and, as such, is able to effect some or all of the actions of LIM protein-like molecules.
- derivative refers to the chemical modification of a nucleic acid encoding HLIM or the encoded HLIM. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group.
- a nucleic acid derivative would encode a polypeptide which retains essential biological characteristics of the natural molecule.
- substantially purified refers to nucleic or amino acid sequences that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated.
- PCR polymerase chain reaction
- hybridization refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
- hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen binds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions.
- the two complementary nucleic acid sequences hydrogen bond in an antiparallel configuration.
- a hybridization complex may be formed in solution (e.g., C 0 t or R graffitit analysis) or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., membranes, filters, chips, pins or glass slides to which cells have been fixed for in situ hybridization).
- complementarity refers to the natural binding of polynucleotides under permissive salt and temperature conditions by base- pairing. For example, for the sequence "A-G-T” binds to the complementary sequence "T-C- A”. Complementarity between two single-stranded molecules may be “partial”, in which only some of the nucleic acids bind, or it may be complete when total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands.
- a partially complementary sequence is one that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid; it is referred to using the functional term "substantially homologous.”
- the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization and the like) under conditions of low stringency.
- a substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence or probe to the target sequence under conditions of low stringency.
- low stringency conditions are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
- the absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence.
- stringent conditions is the “stringency” which occurs within a range from about Tm-5°C (5°C below the melting temperature (Tm) of the probe) to about 20°C to 25°C below Tm.
- Tm melting temperature
- the stringency of hybridization may be altered in order to identify or detect identical or related polynucleotide sequences.
- antisense refers to nucleotide sequences which are complementary to a specific DNA or RNA sequence.
- antisense strand is used in reference to a nucleic acid strand that is complementary to the “sense” strand.
- Antisense molecules may be produced by any method, including synthesis by ligating the gene(s) of interest in a reverse orientation to a viral promoter which permits the synthesis of a complementary strand. Once introduced into a cell, this transcribed strand combines with natural sequences produced by the cell to form duplexes. These duplexes then block either the further transcription or translation. In this manner, mutant phenotypes may be generated.
- the designation “negative” is sometimes used in reference to the antisense strand, and "positive” is sometimes used in reference to the sense strand.
- portion refers to fragments of that protein.
- the fragments may range in size from four amino acid residues to the entire amino acid sequence minus one amino acid.
- a protein "comprising at least a portion of the amino acid sequence of SEQ ID NO: 1" encompasses the full-length human HLIM-1 and fragments thereof.
- Transformation describes a process by which exogenous DNA enters and changes a recipient cell. It may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment.
- Such "transformed” cells include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. They also include cells which transiently express the inserted DNA or RNA for limited periods of time.
- antigenic determinant refers to that portion of a molecule that makes contact with a particular antibody (i.e., an epitope).
- a protein or fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to a given region or three-dimensional structure on the protein; these regions or structures are referred to as antigenic determinants.
- An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
- telomere binding in reference to the interaction of an antibody and a protein or peptide, mean that the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the protein; in other words, the antibody is recognizing and binding to a specific protein structure rather than to proteins in general. For example, if an antibody is specific for epitope "A”, the presence of a protein containing epitope A (or free, unlabeled A) in a reaction containing labeled "A” and the antibody will reduce the amount of labeled A bound to the antibody.
- a biological sample suspected of containing nucleic acid encoding HLIM or fragments thereof may comprise a cell, chromosomes isolated from a cell (e.g., a spread of metaphase chromosomes), genomic DNA (in solution or bound to a solid support such as for Southern analysis), RNA (in solution or bound to a solid support such as for northern analysis), cDNA (in solution or bound to a solid support), an extract from cells or a tissue, and the like.
- correlates with expression of a polynucleotide indicates that the detection of the presence of ribonucleic acid that is similar to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 by northern analysis is indicative of the presence of mRNA encoding HLIM in a sample and thereby correlates with expression of the transcript from the polynucleotide encoding the protein.
- “Alterations” in the polynucleotide of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO: 6, as used herein, comprise any alteration in the sequence of polynucleotides encoding HLIM including deletions, insertions, and point mutations that may be detected using hybridization assays.
- alterations to the genomic DNA sequence which encodes HLIM e.g., by alterations in the pattern of restriction fragment length polymorphisms capable of hybridizing to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6), the inability of a selected fragment of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 to hybridize to a sample of genomic DNA (e.g., using allele-specific oligonucleotide probes), and improper or unexpected hybridization, such as hybridization to a locus other than the normal chromosomal locus for the polynucleotide sequence encoding HLIM (e.g., using fluorescent in situ hybridization (FISH) to metaphase chromosomes spreads).
- FISH fluorescent in situ hybridization
- the term "antibody” refers to intact molecules as well as fragments thereof, such as Fa, F(ab') 2 , and Fv, which are capable of binding the epitopic determinant.
- Antibodies that bind HLIM polypeptides can be prepared using intact polypeptides or fragments containing small peptides of interest as the immunizing antigen.
- the polypeptide or peptide used to immunize an animal can be derived from translated RNA or synthesized chemically, and can be conjugated to a carrier protein, if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin and thyroglobulin. The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).
- humanized antibody refers to antibody molecules in which amino acids have been replaced in the non-antigen binding regions in order to more closely resemble a human antibody, while still retaining the original binding ability.
- the invention is based on the discovery of three novel human LIM proteins (HLIM-1, HLIM-2, and HLIM-3, collectively referred to as HLIM), the polynucleotides encoding HLIM, and the use of these compositions for the diagnosis, prevention, or treatment of cancer, arteriosclerosis, or other diseases or conditions relating to abnormal regulation of cell growth.
- HLIM-1 novel human LIM proteins
- HLIM-2 novel human LIM proteins
- HLIM-3 HLIM-3
- Nucleic acids encoding the human HLIM-1 of the present invention were first identified in Incyte Clone 305288 from an adult heart tissue cDNA library (HEARNOT01) through a computer-generated search for amino acid sequence alignments.
- a consensus sequence, SEQ ID NO:2 was derived from the following overlapping and/or extended nucleic acid sequences (cDNA library from which derived): Incyte Clones 305288, 305719, 305804, 307231 (HEARNOT01); 532714 (BRATNOT03); 963636 (BRSTTUT03); 1004370 (BRSTNOT03); 1266381 (BRAINOT09); and 1436648 (PANCNOT08).
- Nucleic acids encoding the human HLIM-2 of the present invention were first identified in Incyte Clone 267324 from an hNT2 cell line cDNA library (HNT2NOT01) through a computer-generated search for amino acid sequence alignments.
- A. consensus sequence, SEQ ID NO:4, was derived from the following overlapping and/or extended nucleic acid sequences (cDNA library from which derived): Incyte Clones 970132 (MUSCNOT02); 778312 (COLNNOT05); 1291737 (PGANNOT03); 1504324 (BRAITUT07); 922412 (RATRNOT02); 1349314 (LATRTUT02); and 267324 (HNT2NOT01).
- Nucleic acids encoding the human HLIM-3 of the present invention were first identified in Incyte Clone 1429619, from an ileum tissue cDNA library (SINTBST01) through a computer-generated search for amino acid sequence alignments.
- a consensus sequence, SEQ ID NO:6, was derived from the following overlapping and/or extended nucleic acid sequences (cDNA library from which derived): Incyte Clones 1429619
- the invention encompasses the novel human LIM protein HLIM- 1, a polypeptide comprising the amino acid sequence of SEQ ID NO:l, as shown in Figures 1A and IB.
- HLIM-1 is 193 amino acids in length and contains a potential nuclear localization motif comprising K ⁇ 4 I 65 Y 66 G 67 P 68 K 69 .
- HLIM-1 contains a potential protein kinase C phosphorylation site at T, 59 and a potential tyrosine kinase phosphorylation site at Y, 21 .
- HLIM-1 has chemical and structural homology with human CRP (Gl 118161; SEQ ID NO:7) human MLP (Gl 1234841; SEQ ID NO:8), and rat smLIM (Gl 1314351; SEQ ID NO:9).
- HLIM-1 shares 81%, 67%, and 97% identity with human CRP, human MLP and rat smLIM, respectively.
- HLIM and rat smLIM have similar hydrophobicity plots.
- HLIM-1 contains two LIM domains spanning positions C 10 to C 61 and C l l9 to C 170 .
- HLIM-1 is abundantly expressed in heart and bladder tissue CDNA libraries, which suggests a specific role in smooth muscle cell and possibly cardiac muscle cell function.
- HLIM-1 is found in several fetal tissue libraries, which suggests a developmental role for this molecule. Expression of HLIM-1 appears to decrease in tumor associated tissues; for instance, HLIM-1 expression is decreased in 20-fold in heart myoma tumor tissue as compared to normal heart tissue.
- the invention encompasses the novel human LIM protein HLIM-2, a polypeptide comprising the amino acid sequence of SEQ ID NO:3, as shown in Figures 2A, 2B, 2C and 2D.
- HLIM-2 is 329 amino acids in length.
- HLIM-2 has chemical and structural homology with rat CLP36 (Gl 1020151 ; SEQ ID NO:3
- HLIM-2 and CLP36 share 88%> identity, whereas HLIM-2 and RIL share 44 % identity.
- HLIM-2 and CLP36 have rather similar hydrophobicity plots.
- HLIM-2 contains a single LIM domain, defined by amino acids C 260 , C 263 , H 280 , C 283 , C 286 , C 289 , C 307 and H 310 , which is precisely conserved in CLP36 and RIL.
- Northern analysis (not shown) reveals the expression of this sequence in 222 cDNA libraries prepared from a wide variety of tissues, with highest abundance in breast, ovary, uterus, lung, heart, skeletal muscle, and synovium.
- the expression of sequences encoding HLIM-2 appears to be diminished in tumor-associated tissues. Only one of the 40 libraries which express the highest abundance of HLIM-2 are tumor-associated. In particular, tumor-associated tissue from breast and lung contain significantly less HLIM-2 mRNA than normal breast and lung tissue.
- the invention encompasses the novel human LIM protein HLIM-3, a polypeptide comprising the amino acid sequence of SEQ ID NO:5, as shown in Figures 3A, 3B and 3C.
- HLIM-3 is 316 amino acids in length.
- HLIM-3 has chemical and structural homology with rat CLP36 (Gl 1020151; SEQ ID NO: 10) and rat RIL (Gl 887580; SEQ ID NO:l 1).
- HLIM-3 and CLP36 share 53% identity
- HLIM-3 and RIL share 45% identity.
- HLIM-3 and CLP36 have rather similar hydrophobicity plots.
- the single LIM domain of HLIM-3 defined by amino acids C 246 , C 249 , H 266 , C 269 , C 272 , C 275 , C 293 , and H 296 is precisely conserved in CLP36 and RIL.
- Northern analysis shows the expression of this sequence is most abundant in skeletal muscle.
- HLIM-3 encoding sequences are also expressed in cDNA libraries related to smooth and cardiac muscle including heart, ovary, stomach, bladder, prostate, breast, and colon. The expression of sequences encoding HLIM-3 appears to be diminished in tumor-associated tissues; of the 43 libraries which express HLIM-3, only 5 are tumor-associated.
- the invention also encompasses HLIM variants.
- a preferred HLIM variant is one having at least 80%, and more preferably 90%, amino acid sequence similarity to the HLIM amino acid sequence (SEQ ID NO:l, SEQ ID NO:3, or SEQ ID NO:5).
- a most preferred HLIM variant is one having at least 95% amino acid sequence similarity to SEQ ID NO:l, SEQ ID NO:3, or SEQ ID NO:5.
- the invention also encompasses polynucleotides which encode HLIM. Accordingly, any nucleic acid sequence which encodes the amino acid sequence of HLIM can be used to generate recombinant molecules which express HLIM.
- the invention encompasses the polynucleotide comprising the nucleic acid of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 as shown in Figures 1 A, 1 B, 2A, 2B, 2C, 2D, 3 A, 3, 3C and 3D.
- nucleotide sequences encoding HLIM may be produced.
- the invention contemplates each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequence of naturally occurring HLIM, and all such variations are to be considered as being specifically disclosed.
- nucleotide sequences which encode HLIM and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring HLIM under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding HLIM or its derivatives possessing a substantially different codon usage. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic expression host in accordance with the frequency with which particular codons are utilized by the host.
- RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
- the invention also encompasses production of a DNA sequence, or portions thereof, which encode HLIM and its derivatives, entirely by synthetic chemistry.
- the synthetic gene may be inserted into any of the many available DNA vectors and cell systems using reagents that are well known in the art at the time of the filing of this application.
- synthetic chemistry may be used to introduce mutations into a sequence encoding HLIM or any portion thereof.
- polynucleotide sequences that are capable of hybridizing to the claimed nucleotide sequences, and in particular, those shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6, under various conditions of stringency. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe, as taught in immel, A.R. (1987; Methods Enzymol. Vol. 152), and may be used at a defined stringency.
- Altered nucleic acid sequences encoding HLIM which are encompassed by the invention include deletions, insertions, or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent HLIM.
- the encoded protein may also contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent HLIM. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological activity of HLIM is retained.
- negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
- alleles of the gene encoding HLIM are also included within the scope of the present invention.
- an "allele” or “allelic sequence” is an alternative form of the gene which may result from at least one mutation in the nucleic acid sequence. Alleles may result in altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one, or many allelic forms. Common mutational changes which give rise to alleles are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
- Methods for DNA sequencing which are well known and generally available in the art may be used to practice any embodiments of the invention.
- the methods may employ such enzymes as the lenow fragment of DNA polymerase I, Sequenase® (US Biochemical Corp, Cleveland, OH), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, IL), or combinations of recombinant polymerases and proofreading exonucleases such as the ELONGASE Amplification System marketed by Gibco BRL (Gaithersburg, MD).
- the process is automated with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, NV), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, MA) and the ABI 377 DNA sequencers (Perkin Elmer).
- the polynucleotide sequence encoding HLIM may be extended utilizing a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements. For example, one method which may be employed, "restriction-site" PCR, uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, G. (1993) PCR Methods Applic. 2:318-322).
- genomic DNA is first amplified in the presence of primer to linker sequence and a primer specific to the known region.
- the amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one.
- Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
- Inverse PCR may also be used to amplify or extend sequences using divergent primers based on a known region (Triglia, T. et al. (1988) Nucleic Acids Res. 16:8186).
- the primers may be designed using OLIGO® 4.06 Primer Analysis software (National Biosciences Inc., Plymouth, MN), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68°-72° C.
- the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
- Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic. 1 :1 11-119).
- capture PCR involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic. 1 :1 11-119).
- multiple restriction enzyme digestions and ligations may also be used to place an engineered double-stranded sequence into an unknown portion of the DNA molecule before performing PCR.
- Another method which may be used to retrieve unknown sequences is that of Parker, J.D. et al. (1991 ; Nucleic Acids Res. 19:3055-3060). Additionally, one may use PCR, nested primers, and PromoterFinderTM libraries to walk in genomic DNA (Clontech, Palo Alto, CA). This process avoids the need to screen libraries and may be is useful in finding intron/exon junctions.
- libraries that have been size-selected to include larger cDNAs.
- random-primed libraries are preferable in that they will contain more sequences which contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA.
- Genomic libraries may be useful for extension of sequence into the 5' and 3' non-transcribed regulatory regions. Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
- capillary sequencing may employ flowable polymers for electrophoretic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled devise camera.
- Output/light intensity may be converted to electrical signal using appropriate software (e.g. GenotyperTM and Sequence NavigatorTM, Perkin Elmer) and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
- Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA which might be present in limited amounts in a particular sample.
- polynucleotide sequences or fragments thereof which encode HLIM, or fusion proteins or functional equivalents thereof may be used in recombinant DNA molecules to direct expression of HLIM in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express HLIM.
- codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life which is longer than that of a transcript generated from the naturally occurring sequence.
- nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter the HLIM coding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, and/or expression of the gene product.
- DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequence.
- site-directed mutagenesis may be used to insert new restriction sites, alter glycosylation patterns, to change codon preference, to produce splice variants, or other mutations, and so forth.
- a natural, modified, or recombinant polynucleotide encoding HLIM may be ligated to a heterologous sequence to encode a fusion protein.
- a fusion protein may also be engineered to contain a cleavage site located between a HLIM encoding sequence and the heterologous protein sequence, so that HLIM may be cleaved and purified away from the heterologous moiety.
- the coding sequence of HLIM may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers, M.H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).
- the protein itself may be produced using chemical methods to synthesize the HLIM amino acid sequence, or a portion thereof.
- peptide synthesis can be performed using various solid-phase techniques (Roberge, J.Y. et al.
- the newly synthesized peptide may be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, T. (1983) Proteins. Structures and Molecular Principles. WH Freeman and Co., New York, NY).
- the composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra).
- the amino acid sequence of HLIM, or any part thereof may be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins, or any part thereof, to produce a variant polypeptide.
- nucleotide sequence encoding HLIM or functional equivalents may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
- a variety of expression vector/host systems may be utilized to contain and express a HLIM coding sequence. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems.
- microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, T
- control elements are those non-translated regions of the vector—enhancers, promoters, 5' and 3' untranslated regions—which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, LaJolla, CA) or pSportlTM plasmid (Gibco BRL) and ptrp-lac hybrids, and the like may be used.
- inducible promoters such as the hybrid lacZ promoter of the Bluescript® phagemid (Stratagene, LaJolla, CA) or pSportlTM plasmid (Gibco BRL) and ptrp-lac hybrids, and the like may be used.
- the baculovirus polyhedrin promoter may be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO; and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequence encoding HLIM, vectors based on S V40 or EB V may be used with an appropriate selectable marker.
- Promoters or enhancers derived from the genomes of plant cells e.g., heat shock, RUBISCO; and storage protein genes
- plant viruses e.g., viral promoters or leader sequences
- a number of expression vectors may be selected depending upon the use intended for HLIM.
- vectors which direct high level expression of fusion proteins that are readily purified may be used.
- Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as Bluescript® (Stratagene), in which the sequence encoding HLIM may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem.
- pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
- GST glutathione S-transferase
- fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
- Proteins made in such systems may be designed to include heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
- Saccharomyces cerevisiae a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used.
- constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH
- PGH palladium phosphate
- the expression of a sequence encoding HLIM may be driven by any of a number of promoters.
- viral promoters such as the 35S and 19S promoters of CaMV may be used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311).
- plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105).
- These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in a number of generally available reviews (see, for example, Hobbs, S. or Murry, L.E. in McGraw Hill Yearbook of Science and Technology ( 1992) McGraw Hill, New York, NY; pp. 191 - 196.
- An insect system may also be used to express HLIM.
- HLIM Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
- the sequence encoding HLIM may be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of HLIM will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein.
- the recombinant viruses may then be used to infect, for example, S. frugiperda cells or Trichoplusia larvae in which HLIM may be expressed (Engelhard, E.K. et al. (1994) Proc. Nat. Acad. Sci. 91 :3224-3227).
- a number of viral-based expression systems may be utilized.
- a sequence encoding HLIM may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain a viable virus which is capable of expressing HLIM in infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659).
- transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
- RSV Rous sarcoma virus
- Specific initiation signals may also be used to achieve more efficient translation of a sequence encoding HLIM. Such signals include the ATG initiation codon and adjacent sequences. In cases where sequences encoding HLIM, its initiation codon, and upstream sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers which are appropriate for the particular cell system which is used, such as those described in the literature (Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).
- a host cell strain may be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired fashion.
- modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
- Post-translational processing which cleaves a "prepro" form of the protein may also be used to facilitate correct insertion, folding and/or function.
- Different host cells such as CHO, HeLa, MDCK, HEK293, and WI38, which have specific cellular machinery and characteristic mechanisms for such post-translational activities, may be chosen to ensure the correct modification and processing of the foreign protein.
- cell lines which stably express HLIM may be transformed using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or separate vector. Following the introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
- the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be proliferated using tissue culture techniques appropriate to the cell type.
- any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell 11 :223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) genes which can be employed in tk " or aprt cells, respectively. Also, antimetabolite, antibiotic or herbicide resistance can be used as the basis for selection; for example, dhfr which confers resistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
- npt which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14) and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra). Additional selectable genes have been described, for example, trpB, which allows cells to utilize indole in place of tryptophan, or hisD, which allows cells to utilize histinol in place of histidine (Hartman, S.C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci.
- marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be confirmed.
- sequence encoding HLIM is inserted within a marker gene sequence
- recombinant cells containing sequences encoding HLIM can be identified by the absence of marker gene function.
- a marker gene can be placed in tandem with a sequence encoding HLIM under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
- host cells which contain the coding sequence for HLIM and express HLIM may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of the nucleic acid or protein.
- the presence of the polynucleotide sequence encoding HLIM can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or portions or fragments of polynucleotides encoding HLIM.
- Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the HLIM-encoding sequence to detect transformants containing DNA or RNA encoding HLIM.
- DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/
- oligonucleotides or “oligomers” refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides, which can be used as a probe or amplimer.
- Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
- ELISA enzyme-linked immunosorbent assay
- RIA radioimmunoassay
- FACS fluorescence activated cell sorting
- Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding HLIM include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide.
- the sequence encoding HLIM, or any portion of it may be cloned into a vector for the production of an mRNA probe.
- RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
- T7, T3, or SP6 RNA polymerase
- Suitable reporter molecules or labels include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
- Host cells transformed with a nucleotide sequence encoding HLIM may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
- the protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
- expression vectors containing polynucleotides which encode HLIM may be designed to contain signal sequences which direct secretion of HLIM through a prokaryotic or eukaryotic cell membrane.
- Other recombinant constructions may be used to join sequences encoding HLIM to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins.
- Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA).
- metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals
- protein A domains that allow purification on immobilized immunoglobulin
- the domain utilized in the FLAGS extension/affinity purification system Immunex Corp., Seattle, WA.
- cleavable linker sequences such as those specific for Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and HLIM may be used to facilitate purification.
- One such expression vector provides for expression of a fusion protein containing HLIM and a nucleic acid encoding 6 histidine residues preceding a thioredoxin or an enterokinase cleavage site.
- the histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography) as described in Porath, J. et al. (1992, Prot. Exp. Purif. 3: 263-281) while the enterokinase cleavage site provides a means for purifying HLIM from the fusion protein.
- IMIAC immobilized metal ion affinity chromatography
- fragments of HLIM may be produced by direct peptide synthesis using solid-phase techniques (cf Stewart et al. (1969) Solid-Phase Peptide Synthesis. W.H. Freeman Co., San Francisco, CA; Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer). Various fragments of HLIM may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
- HLIM or fragments thereof may be used for therapeutic purposes.
- HLIM sequences disclosed herein are based in part on the chemical and structural homology among HLIM- 1 , human MLP and rat smLIM, and among HLIM-2, HLIM-3, rat CLP36 and rat RIL. Furthermore, the expression patterns of HLIM suggest a role in promoting or maintaining normal tissue growth and differentiation. HLIM-1 and HLIM-3 are expressed primarily in muscle tissues, whereas HLIM-2 is expressed in a wide variety of tissues. Since expression of HLIM appears to be negatively correlated with cancer ( Figures 12 and 13, and as discussed above), vectors containing the nucleic acid sequence encoding HLIM may be used to promote differentiation and to restore tumor cells to a normal phenotype .
- HLIM activity as a novel approach to cancer treatment may be especially useful in combination therapy with other, conventional chemotherapeutic agents.
- Such combinations of therapeutic agents having different cellular mechanisms of action often have synergistic effects allowing the use of lower effective doses of each agent and lessening side effects.
- vectors expressing antisense, and antagonists or inhibitors of the protein may be used to induce proliferation or regeneration of cells, tissues and organs which are not readily regenerated. Regeneration and development of nerve, pancreatic, epithelial, etc. tissue will involve supplying various molecules including HLIM antagonists in drug regimes that allow cell division to occur. Small amounts of new functional tissue have the capacity to greatly improve the quality of life for victims of accidents or degenerative diseases. Furthermore, HLIM antagonists may be useful in stimulating skin production in vitro or ex vivo for use in skin grafts.
- antagonists which block or modulate the effect of HLIM may be used in those situations where such inhibition is therapeutically desirable.
- Such antagonists or inhibitors may be produced using methods which are generally known in the art, and include particularly the use of purified HLIM to produce antibodies or to screen libraries of pharmaceutical agents for those which specifically bind HLIM.
- antibodies which are specific for HLIM may be used directly as an antagonist, or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express HLIM.
- the antibodies may be generated using methods that are well known in the art.
- Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, and fragments produced by a Fab expression library.
- Neutralizing antibodies are especially preferred for therapeutic use.
- various hosts including goats, rabbits, rats, mice, humans, and others, may be immunized by injection with HLIM or any fragment or oligopeptide thereof which has immunogenic properties.
- various adjuvants may be used to increase immunological response.
- adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
- BCG Bacilli Calmette-Guerin
- Corynebacterium parvum are especially preferable.
- the peptides, fragments, or oligopeptides used to induce antibodies to HLIM have an amino acid sequence consisting of at least five amino acids, and more preferably at least 10 amino acids. It is also preferable that they are identical to a portion of the amino acid sequence of the natural protein, and they may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of HLIM amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule. Monoclonal antibodies to HLIM may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
- chimeric antibodies the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison et al. (1984) Proc. Natl. Acad. Sci. 81 :6851-6855; Neuberger et al. (1984) Nature 312:604-608; Takeda et al. (1985) Nature 314:452-454).
- techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce HLIM-specific single chain antibodies.
- Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobin libraries (Burton D.R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).
- Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al. (1991) Nature 349:293-299).
- Antibody fragments which contain specific binding sites for HLIM may also be generated.
- fragments include, but are not limited to, the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
- Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al. (1989) Science 256:1275-1281).
- immunoassays may be used for screening to identify antibodies having the desired specificity.
- Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
- Such immunoassays typically involve the measurement of complex formation between HLIM and its specific antibody.
- a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering HLIM epitopes is preferred, but a competitive binding assay may also be employed (Maddox, supra).
- the polynucleotides encoding HLIM, or any fragment thereof, or antisense sequences may be used for therapeutic purposes.
- antisense to the polynucleotide encoding HLIM may be used in situations in which it would be desirable to block the synthesis of the protein.
- cells may be transformed with sequences complementary to polynucleotides encoding HLIM.
- antisense sequences may be used to modulate HLIM activity, or to achieve regulation of gene function.
- sense or antisense oligomers or larger fragments can be designed from various locations along the coding or control regions of sequences encoding HLIM.
- Expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population. Methods which are well known to those skilled in the art can be used to construct recombinant vectors which will express antisense polynucleotides of the gene encoding HLIM. These techniques are described both in Sambrook et al. (supra) and in Ausubel et al. (supra).
- Genes encoding HLIM can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide or fragment thereof which encodes HLIM. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until all copies are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector and even longer if appropriate replication elements are part of the vector system.
- modifications of gene expression can be obtained by designing antisense molecules, DNA, RNA, or PNA, to the control regions of the gene encoding HLIM, i.e., the promoters, enhancers, and introns. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J.E. et al.
- the antisense molecules may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
- Ribozymes enzymatic RNA molecules
- Ribozymes may also be used to catalyze the specific cleavage of RNA.
- the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples which may be used include engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding HLIM.
- ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
- Antisense molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding HLIM. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells, or tissues.
- RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
- vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection and by liposome injections may be achieved using methods which are well known in the art.
- compositions may consist of HLIM, antibodies to HLIM, mimetics, agonists, antagonists, or inhibitors of HLIM.
- the compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
- the compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones.
- compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
- these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, PA).
- compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
- Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
- compositions for oral use can be obtained through combination of active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
- Suitable excipients are carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; gums including arabic and tragacanth; and proteins such as gelatin and collagen.
- disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
- Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
- suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
- Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
- compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
- Push-fit capsules can contain active ingredients mixed with a filler or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
- the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
- compositions suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiologically buffered saline.
- Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
- suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
- Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
- the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
- suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
- penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
- compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
- the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
- the preferred preparation may be a lyophilized powder which may contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use. After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition.
- compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.
- the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs.
- the animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
- a therapeutically effective dose refers to that amount of active ingredient, for example HLIM or fragments thereof, antibodies of HLIM, agonists, antagonists or inhibitors of HLIM, which ameliorates the symptoms or condition.
- Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50 ED50.
- Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
- Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
- Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation. Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
- antibodies which specifically bind HLIM may be used for the diagnosis of conditions or diseases characterized by expression of HLIM, or in assays to monitor patients being treated with HLIM, agonists, antagonists or inhibitors.
- the antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics.
- Diagnostic assays for HLIM include methods which utilize the antibody and a label to detect HLIM in human body fluids or extracts of cells or tissues.
- the antibodies may be used with or without modification, and may be labeled by joining them, either covalently or non-covalently, with a reporter molecule.
- a wide variety of reporter molecules which are known in the art may be used, several of which are described above.
- HLIM human-derived neurotrophic factor
- ELISA ELISA
- RIA RIA
- FACS FACS-activated HLIM
- normal or standard values for HLIM expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to HLIM under conditions suitable for complex formation The amount of standard complex formation may be quantified by various methods, but preferably by photometric, means. Quantities of HLIM expressed in subject, control and disease, samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
- the polynucleotides encoding HLIM may be used for diagnostic purposes.
- the polynucleotides which may be used include oligonucleotide sequences, antisense RNA and DNA molecules, and PNAs.
- the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of HLIM may be correlated with disease.
- the diagnostic assay may be used to distinguish between absence, presence, and excess expression of HLIM, and to monitor regulation of HLIM levels during therapeutic intervention.
- hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding HLIM or closely related molecules, may be used to identify nucleic acid sequences which encode HLIM.
- the specificity of the probe whether it is made from a highly specific region, e.g., 10 unique nucleotides in the 5' regulatory region, or a less specific region, e.g., especially in the 3' coding region, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low) will determine whether the probe identifies only naturally occurring sequences encoding HLIM, alleles, or related sequences.
- Probes may also be used for the detection of related sequences, and should preferably contain at least 50% of the nucleotides from any of the HLIM encoding sequences.
- the hybridization probes of the subject invention may be DNA or RNA and derived from the nucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 or from genomic sequence including promoter, enhancer elements, and introns of the naturally occurring HLIM.
- Means for producing specific hybridization probes for DNAs encoding HLIM include the cloning of nucleic acid sequences encoding HLIM or HLIM derivatives into vectors for the production of mRNA probes.
- Such vectors are known in the art, commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
- Hybridization probes may be labeled by a variety of reporter groups, for example, radionuclides such as 32P or 35S, or enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
- Polynucleotide sequences encoding HLIM may be used for the diagnosis of conditions or diseases which are associated with expression of HLIM. Examples of such conditions or diseases include cancers of the heart, breast, colon, and prostate, and diseases associated with proliferation of smooth muscle cells such as arteriosclerosis.
- the polynucleotide sequences encoding HLIM may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; or in dip stick, pin, ELISA or chip assays utilizing fluids or tissues from patient biopsies to detect altered HLIM expression. Such qualitative or quantitative methods are well known in the art.
- the nucleotide sequences encoding HLIM may be useful in assays that detect activation or induction of various cancers, particularly those mentioned above .
- the nucleotide sequence encoding HLIM may be labeled by standard methods, and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value.
- nucleotide sequence has hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding HLIM in the sample indicates the presence of the associated disease.
- assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
- a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, which encodes HLIM, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for disease. Deviation between standard and subject values is used to establish the presence of disease.
- hybridization assays may be repeated on a regular basis to evaluate whether the level of expression in the patient begins to approximate that which is observed in the normal patient.
- the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
- oligonucleotides encoding HLIM may involve the use of PCR. Such oligomers may be chemically synthesized, generated enzymatically, or produced from a recombinant source.
- Oligomers will preferably consist of two nucleotide sequences, one with sense orientation (5'->3') and another with antisense (3' ⁇ -5'), employed under optimized conditions for identification of a specific gene or condition.
- the same two oligomers, nested sets of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences.
- Methods which may also be used to quantitate the expression of HLIM include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated (Melby, P.C. et al. (1993) J. Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236).
- the speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
- the nucleic acid sequence which encodes may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
- HLIM may also be used to generate hybridization probes which are useful for mapping the naturally occurring genomic sequence.
- the sequence may be mapped to a particular chromosome or to a specific region of the chromosome using well known techniques .
- Such techniques include FISH, FACS, or artificial chromosome constructions, such as yeast 5 artificial chromosomes, bacterial artificial chromosomes, bacterial PI constructions or single chromosome cDNA libraries as reviewed in Price, CM. (1993) Blood Rev. 7:127-134, and Trask, B.J. (1991) Trends Genet. 7: 149-154.
- FISH FISH (as described in Verma et al. (1988) Human Chromosomes: A Manual of Basic Techniques. Pergamon Press, New York, NY) may be correlated with other physical ⁇ o chromosome mapping techniques and genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265: 198 If). Correlation between the location of the gene encoding HLIM on a physical chromosomal map and a specific disease , or predisposition to a specific disease, may help delimit the region of DNA associated with that genetic disease.
- the nucleotide sequences of the subject invention may be used to detect
- In situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a
- any sequences mapping to that area may represent associated or regulatory genes for further investigation.
- the nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier, or affected individuals.
- HLIM its catalytic or immunogenic
- fragments or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques.
- the fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly.
- the formation of binding complexes, between HLIM and the agent being tested, may be measured.
- Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO84/03564. In this method, as applied to HLIM large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with HLIM, or fragments thereof, and washed.
- Bound HLIM is then detected by methods well known in the art. Purified HLIM can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
- nucleotide sequences which encode HLIM may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
- the HEARNOT01 library was constructed from heart tissue obtained from a 56 year- old Caucasian male (Lot No. HAL 194, International Institute for the Advancement of Medicine (HAM), Exton, PA). The frozen tissue was homogenized and lysed using a Brinkmann Homogenizer
- the mRNA was then isolated using the Qiagen Oligotex kit (Qiagen, Inc., Chatsworth, CA) and used to construct the cDNA library.
- First strand cDNA synthesis was accomplished using an oligo d(T) primer/linker which also contained an Xhol restriction site.
- Second strand synthesis was performed using a combination of DNA polymerase I, coli ligase and RNase H, followed by the addition of an EcoRI adaptor to the blunt ended cDNA.
- the EcoRI adapted, double-stranded cDNA was then digested with Xhol restriction enzyme and fractionated to obtain sequences which exceeded 800 bp in size.
- the cDNAs were inserted into the LambdaZap ® vector system (Stratagene); then the vector which contained the pBluescript phagemid (Stratagene) was transformed into E. con ' host cells strain XLl-BlueMRFTM (Stratagene).
- the phagemid forms of individual cDNA clones were obtained by the in vivo excision process. Enzymes from both pBluescript and a cotransformed fl helper phage nicked the DNA, initiated new DNA synthesis, and created the smaller, single-stranded circular phagemid molecules which contained the cDNA insert. The phagemid DNA was released, purified, and used to reinfect fresh host cells (SOLR, Stratagene). Presence of the phagemid which contained the gene for ⁇ -lactamase allowed transformed bacteria to grow on medium containing ampicillin. HNT2NOT01
- the hNT2 cell line exhibits characteristics of a committed neuronal precursor cell which is still at an early stage of development.
- the HNT2NOT01 cDNA library prepared from this untreated cell line was obtained from Stratagene (Cat. No. 937230).
- the cDNA library was constructed by essentially the following procedure. cDNAs were primed using oligo d(T) and size fractionated to isolate fragments of 500 bp and larger. Synthetic adapter oligonucleotides were ligated onto the cDNA molecules enabling them to be inserted into the Uni-ZAPTM vector system (Stratagene). The quality of the cDNA library was screened using DNA probes, and then, the pBluescript phagemid (Stratagene) was excised. Subsequently, the custom-constructed library phage particles were infected into E. coli host strain XL1- Blue® (Stratagene). SINTBST01
- the SINTBST01 cDNA library was constructed from Crohn s disease affected small intestine tissue obtained from an 18-year-old Caucasian female (specimen #0256A; Mayo Clinic, Rochester, MN). This tissue was associated with cDNA library COLNNOT19 which was microscopically normal colon tissue from the same patient.
- Crohn's disease was identified involving 15 cm of the small bowel. The cecum and appendix were unremarkable, and the margins were uninvolved.
- the patient was treated with Priloxec® (omeprazole for abdominal pain; Astra/Merck Group of Merck & Co.,
- Ileum mRNA was extracted from the tissue and purified essentially as described for the HEARNOT01 library. The mRNA was handled according to the recommended protocols in the Superscript Plasmid System for cDNA Synthesis and Plasmid Cloning (Cat. No. 18248-013, Gibco/BRL).
- the commercial plasmid pSPORT 1 (Gibco/BRL) was digested with EcoR I restriction enzyme (New England Biolabs, Beverley, MA). The overhanging ends of the plasmid were filled in using Klenow enzyme (New England Biolabs) and 2'-deoxynucleotide 5'-triphosphates (dNTPs). The plasmid was self-ligated and transformed into the bacterial host, E. coli strain JM 109. An intermediate plasmid produced by the bacteria failed to digest with EcoR I confirming the desired loss of the EcoR I restriction site.
- This intermediate plasmid (pSPORT 1- ⁇ RI) was then digested with Hind HI restriction enzyme (New England Biolabs) and the overhang was filled in with Klenow and dNTPs. A 10-mer linker of sequence 5'...CGGAATTCCG...3' was phosphorylated and ligated onto the blunt ends. The product of the ligation reaction was digested with EcoR I and self- ligated. Following transformation into JM109 host cells, plasmids were isolated and screened 5 for the digestibility with EcoR I but not with Hind III. A single colony which met this criteria was designated pINCY 1. The plasmid produced by this colony was sequenced and found to contain several copies of the 10-mer linker. These extra linkers did not present a problem as they were eliminated when the vector was prepared for cloning.
- the plasmid was tested for its ability to incorporate cDNAs from a library prepared o using Not I and EcoR I restriction enzymes. Several clones were sequenced and a single clone containing an insert of approximately 0.8 kb was selected to prepare a large quantity of the plasmid for library production. After digestion with Not I and EcoR I, the plasmid and the cDNA insert were isolated on an agarose gel and the vector was purified on a QIAQuick (Qiagen, Inc.) column for use in library construction. 5 cDNAs were fractionated on a Sepharose CL4B column (Cat. No. 275105-01 ,
- Plasmid or phagemid DNA was released from cells and purified using the Miniprep
- Kit (Cat. No. 77468; Advanced Genetic Technologies Corporation, Gaithersburg MD). This kit consists of a 96 well block with reagents for 960 purifications. The recommended protocol was employed except for the following changes: 1) the 96 wells were each filled with only 1 ml of sterile Terrific Broth (Cat. No.
- MAGIC MINIPREPSTM DNA Purification System Cat. No. A7100, Promega
- QIAwellTM-8 Plasmid QIAwell PLUS DNA and QIAwell ULTRA DNA Purification Systems (Qiagen, Inc.).
- the cDNAs were sequenced by the method of Sanger F. and A.R. Coulson (1975; J.
- Each cDNA was compared to sequences in GenBank using a search algorithm developed by Applied Biosystems and incorporated into the INHERIT TM 670 sequence analysis system.
- Pattern Specification Language TRW Inc, Los Angeles, CA
- the three parameters that determine how the sequence comparisons run were window size, window offset, and error tolerance.
- the DNA database was searched for sequences containing regions of homology to the query sequence, and the appropriate sequences were scored with an initial value. Subsequently, these homologous regions were examined using dot matrix homology plots to distinguish regions of homology from chance matches. Smith- Waterman alignments were used to display the results of the homology search.
- Peptide and protein sequence homologies were ascertained using the INHERIT- 670 sequence analysis system using the methods similar to those used in DNA sequence homologies. Pattern Specification Language and parameter windows were used to search protein databases for sequences containing regions of homology which were scored with an initial value. Dot-matrix homology plots were examined to distinguish regions of significant homology from chance matches.
- BLAST which stands for Basic Local Alignment Search Tool (Altschul, S.F. (1993) J. Mol. Evol. 36:290-300; Altschul et al. (1990) J. Mol. Biol. 215:403-410), was used to search for local sequence alignments.
- BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying homologs. BLAST is useful for matches which do not contain gaps.
- the fundamental unit of BLAST algorithm output is the High-scoring Segment Pair (HSP).
- An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user.
- the BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance.
- the parameter E establishes the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPs) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output. IV Northern Analysis
- Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound (Sambrook et al., supra).
- Analogous computer techniques using BLAST Altschul, S.F. 1993 and 1990, supra are used to search for identical or related molecules in nucleotide databases such as GenBank or the LIFESEQTM database (Incyte Pharmaceuticals). This analysis is much faster than multiple, membrane-based hybridizations.
- the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or homologous.
- the basis of the search is the product score which is defined as: % sequence identity x % maximum BLAST score 100
- the product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1 -2% error; and at 70, the match will be exact. Homologous molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.
- Full length HLIM-encoding nucleic acid sequence (SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO: 6) is used to design oligonucleotide primers for extending a partial nucleotide sequence to full length or for obtaining 5' or 3', intron or other control sequences from genomic libraries.
- One primer is synthesized to initiate extension in the antisense direction (XLR) and the other is synthesized to extend sequence in the sense direction (XLF).
- Primers are used to facilitate the extension of the known sequence "outward" generating amplicons containing new, unknown nucleotide sequence for the region of interest.
- the initial primers are designed from the cDNA using OLIGO 4.06 (National Biosciences), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68° -72° C Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations is avoided.
- the original, selected cDNA libraries, or a human genomic library are used to extend the sequence; the latter is most useful to obtain 5' upstream regions. If more extension is necessary or desired, additional sets of primers are designed to further extend the known region.
- PCR is performed using the Peltier Thermal Cycler (PTC200; M.J. Research, Watertown, MA) and the following parameters:
- Step 1 94° C for 1 min (initial denaturation)
- Step 3 68° C for 6 min
- Step 4 94° C for 15 sec
- Step 5 65° C for 1 min
- Step 6 68° C for 7 min
- Step 7 Repeat step 4-6 for 15 additional cycles
- Step 8 94° C for 15 sec
- Step 1 Repeat step 8-10 for 12 cycles
- a 5-10 ⁇ aliquot of the reaction mixture is analyzed by electrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gel to determine which reactions were successful in extending the sequence. Bands thought to contain the largest products are selected and removed from the gel. Further purification involves using a commercial gel extraction method such as QIAQUICK (Qiagen Inc.). After recovery of the DNA, Klenow enzyme is used to trim single-stranded, nucleotide overhangs creating blunt ends which facilitate religation and cloning.
- QIAQUICK Qiagen Inc.
- Step 1 94° C for 60 sec
- Step 2 94° C for 20 sec
- Step 3 55° C for 30 sec
- Step 4 72° C for 90 sec
- Step 5 Repeat steps 2-4 for an additional 29 cycles
- Hybridization probes derived from SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base-pairs, is specifically described, essentially the same procedure is used with larger cDNA fragments.
- Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmol of each oligomer and 250 mCi of [ ⁇ - 32 P] adenosine triphosphate (Amersham) and T4 polynucleotide kinase (DuPont NEN ® , Boston, MA).
- the labeled oligonucleotides are substantially purified with Sephadex G-25 superfine resin column (Pharmacia & Upjohn).
- a portion containing 10 7 counts per minute of each of the sense and antisense oligonucleotides is used in a typical membrane based hybridization analysis of human genomic DNA digested with one of the following endonucleases (Ase I, Bgl II, Eco Rl, Pst I, Xba 1 , or Pvu II; DuPont NEN ® ).
- Antisense molecules to the HLIM-encoding sequence, or any part thereof, is used to inhibit in vivo or in vitro expression of naturally occurring HLIM. Although use of antisense oligonucleotides, comprising about 20 base-pairs, is specifically described, essentially the same procedure is used with larger cDNA fragments.
- An oligonucleotide based on the coding sequences of HLIM, as shown in Figures 1 A, IB, 2A, 2B, 2C, 2D, 3 A, 3B, 3C and 3D, is used to inhibit expression of naturally occurring HLIM.
- the complementary oligonucleotide is designed from the most unique 5' sequence as shown in Figures 1A, IB, 2 A, 2B, 2C, 2D, 3 A, 3B, 3C and 3D and used either to inhibit transcription by preventing promoter binding to the upstream nontranslated sequence or translation of an HLIM-encoding transcript by preventing the ribosome from binding.
- an effective antisense oligonucleotide includes any 15-20 nucleotides spanning the region which translates into the signal or 5' coding sequence of the polypeptide as shown in Figures 1 A, IB, 2A, 2B, 2C, 2D, 3A, 3B, 3C and 3D. VIII Expression of HLIM
- HLIM HLIM expression is accomplished by subcloning the cDNAs into appropriate vectors and transforming the vectors into host cells.
- the cloning vector, pSport previously used for the generation of the cDNA library is used to express HLIM in E. coli. Upstream of the cloning site, this vector contains a promoter for ⁇ -galactosidase, followed by sequence containing the amino-terminal Met, and the subsequent seven residues of ⁇ -galactosidase. Immediately following these eight residues is a bacteriophage promoter useful for transcription and a linker containing a number of unique restriction sites.
- the binding of Zn 2 " to HLIM is assayed by monitoring the resulting changes in enthalpy (heat production or absorption) in an isothermal titration microcalorimeter (Micro- Cal Inc., Northampton, MA). Titration microcalorimetry measurements do not require labeling of the ligand or receptor molecules; detection is based solely on the intrinsic change in the heat of enthalpy upon binding.
- Multiple computer-controlled injections of a known volume of ZnCl 2 solution are directed into a thermally-controlled chamber containing HLIM. The change in enthalpy after each injection is plotted against the number of injections, producing a binding isotherm.
- the volumes and concentrations of the injected ZnCl 2 solution and of the HLIM solution are used along with the binding isotherm to calculate values for the number, affinity, and association constant of HLIM with the Zn 2+ ligand.
- HLIM that is substantially purified using PAGE electrophoresis (Sambrook, supra), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols.
- the amino acid sequence deduced from SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 is analyzed using DNASTAR software (DNASTAR Inc) to determine regions of high immunogenicity and a corresponding oligopolypeptide is synthesized and used to raise antibodies by means known to those of skill in the art. Selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions, is described by Ausubel et al. (supra), and others. Typically, the oligopeptides are 15 residues in length, synthesized using an Applied
- Naturally occurring or recombinant HLIM is substantially purified by immunoaffinity chromatography using antibodies specific for HLIM.
- An immunoaffinity column is constructed by covalently coupling HLIM antibody to an activated chromatographic resin, such as CnBr-activated Sepharose (Pharmacia & Upjohn). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
- HLIM Media containing HLIM is passed over the immunoaffinity column, and the column is washed under conditions that allow the preferential absorbance of HLIM (e.g., high ionic strength buffers in the presence of detergent).
- the column is eluted under conditions that disrupt antibody/HLIM binding (eg, a buffer of pH 2-3 or a high concentration of a chaotrope, such as urea or thiocyanate ion), and HLIM is collected.
- a chaotrope such as urea or thiocyanate ion
- Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled HLIM, washed and any wells with labeled HLIM complex are assayed. Data obtained using different concentrations of HLIM are used to calculate values for the number, affinity, and association of HLIM with the candidate molecules.
- CAGCAGATAG ACCTCCAGGG CCCGGGGCCG TGGGGCTTCC GCCTCGTGGG CGGCAAGGAC 180
- a substantially purified human LIM protein comprising the amino acid sequence of SEQ ID NO: l or fragments thereof.
- a hybridization probe comprising the polynucleotide sequence of claim 2.
- An isolated and purified polynucleotide sequence comprising SEQ ID NO:2 or variants thereof. 6. A polynucleotide sequence which is complementary to SEQ ID NO:2 or variants thereof.
- a hybridization probe comprising the polynucleotide sequence of claim 6.
- a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a fragment thereof comprising the steps of: a) culturing the host cell of claim 9 under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
- a pharmaceutical composition comprising a substantially purified human LIM protein having an amino acid sequence of SEQ ID NO: l in conjunction with a suitable pharmaceutical carrier.
- a purified antibody which binds specifically to the polypeptide of claim 1.
- a purified agonist which specifically binds to and modulates the activity of the polypeptide of claim 1.
- a purified antagonist which specifically binds to and blocks the activity of the polypeptide of claim 1.
- a method for treating cancer comprising administering to a subject in need of such treatment an effective amount of the pharmaceutical composition of claim 1 1.
- 5 16 A method for detection of polynucleotides encoding the human LIM protein of claim 1 in a biological sample comprising the steps of: a) hybridizing a polynucleotide consisting of SEQ ID NO:2 to nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting said hybridization complex, wherein the presence of said complex o correlates with the presence of a polynucleotide encoding human LIM protein in said biological sample.
- nucleic acid material of the biological sample is amplified by the polymerase chain reaction.
- a substantially purified human LIM protein comprising the amino acid sequence of 5 SEQ ID NO:3 or fragments thereof.
- a hybridization probe comprising the polynucleotide sequence of claim 19.
- a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO: 3 or a fragment thereof comprising the steps of: 0 a) culturing the host cell of claim 26 under conditions suitable for the expression of the polypeptide; and b) recovering the polypeptide from the host cell culture.
- a pharmaceutical composition comprising a substantially purified human LIM protein having an amino acid sequence of SEQ ID NO:3 in conjunction with a suitable
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Abstract
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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US739485 | 1996-10-28 | ||
US08/739,485 US5863898A (en) | 1996-10-29 | 1996-10-29 | Human lim proteins |
PCT/US1997/018717 WO1998018822A2 (fr) | 1996-10-28 | 1997-10-10 | Nouvelles proteines humaines lim |
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Application Number | Title | Priority Date | Filing Date |
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EP97910998A Ceased EP0931095A2 (fr) | 1996-10-28 | 1997-10-10 | Nouvelles proteines humaines lim |
Country Status (5)
Country | Link |
---|---|
US (2) | US5863898A (fr) |
EP (1) | EP0931095A2 (fr) |
AU (1) | AU4824197A (fr) |
CA (1) | CA2272825A1 (fr) |
WO (1) | WO1998018822A2 (fr) |
Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5767262A (en) * | 1996-03-15 | 1998-06-16 | President And Fellows Of Harvard College | Smooth muscle cell LIM protein |
US6514935B1 (en) | 1997-03-14 | 2003-02-04 | President And Fellows Of Harvard | Methods of treating hypertension |
WO2000014231A1 (fr) * | 1998-09-03 | 2000-03-16 | Incyte Pharmaceuticals, Inc. | Homologue des proteines du domaine lim |
US20050037969A1 (en) * | 1999-05-14 | 2005-02-17 | Arbor Vita Corporation | Molecular interactions in hematopoietic cells |
US6942981B1 (en) * | 1999-05-14 | 2005-09-13 | Arbor Vita Corporation | Method of determining interactions with PDZ-domain polypeptides |
US20060148711A1 (en) * | 1999-05-14 | 2006-07-06 | Arbor Vita Corporation | Molecular interactions in neurons |
DE10004857A1 (de) * | 2000-02-03 | 2001-08-16 | Schering Ag | MLP-Gen, Nukleinsäuren, Polypeptide und deren Verwendung |
KR100421464B1 (ko) * | 2000-07-11 | 2004-03-10 | 지성규 | 체내에 흡수되기 용이한 구리-올리고펩티드의 제조방법 |
US20040229298A1 (en) * | 2000-11-11 | 2004-11-18 | Lu Peter S. | Methods and compositions for treating cervical cancer |
AU2002252015A1 (en) * | 2001-02-16 | 2002-09-04 | Arbor Vita Corporation | Pdz domain interactions and lipid rafts |
US6770797B2 (en) | 2001-06-01 | 2004-08-03 | Rhode Island Hospital | Non-Transgenic nonhuman model for Alzheimer's Disease using a AD7c-NTP nucleic acid |
US8415315B2 (en) | 2004-05-06 | 2013-04-09 | University Of Central Florida Research Foundation, Inc. | Methods and compositions for inhibiting the proliferation of cancer cells |
-
1996
- 1996-10-29 US US08/739,485 patent/US5863898A/en not_active Expired - Fee Related
-
1997
- 1997-10-10 CA CA002272825A patent/CA2272825A1/fr not_active Abandoned
- 1997-10-10 AU AU48241/97A patent/AU4824197A/en not_active Abandoned
- 1997-10-10 EP EP97910998A patent/EP0931095A2/fr not_active Ceased
- 1997-10-10 WO PCT/US1997/018717 patent/WO1998018822A2/fr not_active Application Discontinuation
-
2002
- 2002-04-18 US US10/126,099 patent/US20020170079A1/en not_active Abandoned
Non-Patent Citations (1)
Title |
---|
See references of WO9818822A3 * |
Also Published As
Publication number | Publication date |
---|---|
CA2272825A1 (fr) | 1998-05-07 |
WO1998018822A3 (fr) | 1998-07-16 |
AU4824197A (en) | 1998-05-22 |
WO1998018822A2 (fr) | 1998-05-07 |
US5863898A (en) | 1999-01-26 |
US20020170079A1 (en) | 2002-11-14 |
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